The long-term goals of this research are directed at elucidating the mechanisms involved in the biogenesis and assembly of the outer membrane of gram-negative bacteria. It is anticipated that a knowledge of this process will provide a rational basis for the development of new antimicrobial agents and also provide insights into the processes involved in membrane biogenesis and assembly in eukaryotic cells and cell organelles. The specific focus of this research is to define the mechanism of biosynthesis and assembly of enterobacterial common antigen (ECA). ECA is an outer membrane glycolipid that is unique to the Enterobacteriaceae, and it occurs in all bacteria belonging to this family. We have investigated ECA synthesis in Salmonella tymphimurium and Escherichia coli, and both in vivo and in vitro systems have been successfully developed and employed for this purpose. We wish to continue our studies, and the specific aims for the requested period of support are (a) to demonstrate that Fuc4NAc- ManNAcA-GlcNAc-pyrophosphorylundecaprenol is a precursor of ECA heteropolysaccharide chains; (b) to determine the mechanism involved in' the assembly of ECA chains; (c) to determine the mechanism involved in the synthesis of phosphoglyceride-linked ECA chains (ECApG); (d) to determine the mechanism involved in the synthesis of lipopolysaccharide-linked ECA (ECALPS); and (3) to isolate and structurally characterize polyisoprenoid-linked intermediates involved in ECA synthesis. The proposed research will utilize techniques of molecular biology and biochemistry including in vivo pulse-labeling, enzymology, and the isolation and structural characterization of biosynthetic intermediates.